IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings

Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use biochemical and structural approaches to define the molecular determinants by which IstA, a transposase present in the widespread IS21 family of mobile elements, catalyzes efficient DNA transposition. Solution studies show that IstA engages the transposon terminal sequences to form a high-molecular weight complex and promote DNA integration. A 3.4 Å resolution structure of the transposase bound to transposon ends corroborates our biochemical findings and reveals that IstA self-assembles into a highly intertwined tetramer that synapses two supercoiled terminal inverted repeats. The three-dimensional organization of the IstA•DNA cleaved donor complex reveals remarkable similarities with retroviral integrases and classic transposase systems, such as Tn7 and bacteriophage Mu, and provides insights into IS21 transposition.

, and quantification of the bands in the native gel (lower panel). Product of the reaction labeled as supercoiled (sc, blue), linear (lin, red) and relaxed (rlx, yellow) DNA. Molecular weight marker (in kb) indicated as M. This experiment was repeated three independent times. Source data are provided as a Source Data file. b, Integration reactions with the IstA-MBP fusion protein used in fluorescence anisotropy experiments show that the activity of the tagged enzyme is comparable to that of wildtype IstA. Molecular weight marker (in kb) indicated as M. This experiment was repeated three independent times. Source data are provided as a Source Data file.

Supplementary Fig. 3 | Cryo-EM structures of IstA in complex with the isolated right TIR and a stoichiometric mixture of left and right
TIRs. The 2D class averages and different orientations of the 3D reconstructions obtaining without imposing symmetry (C1) indicate that the transposase adopts a similar configuration when bound to the isolated right TIR or a equimolar mix of left and right TIRs. Supplementary Fig. 4 | High-resolution cryo-EM analysis and image processing. a, Representative cryo-EM micrograph of IstA•TIR complexes (from a total of 7,215 images). b, Image processing workflow. c, Fourier Shell Correlation of the final density map. Source data are provided as a Source Data file. d, Unsharpened map showed at two different thresholds (0.0035 and 0.01). The upper and lower regions of the density are particularly flexible. e, Angular distribution plot showing the range of observed particle orientations. f, Local resolution of the unsharpened map (same as panel (d); 0.0035 threshold).

Supplementary Fig. 5 | Detailed views relevant regions of the map and atomic
model. a, Protein region comprising residues 228-245 in the DDE domain of the catalytic subunits (density map is contoured at a threshold value of 0.025 in all panels). b, CS1 DNA region (DA10-DT51 and complementary DG16-DC45 nucleotides). c, Interface between upper and lower chains showing protein-protein interaction details. d, IstA active site. Catalytic triad and some key elements implicated in stabilizing the flipped 5' overhang are labelled. e, Specific recognition of the conserved DNA sequence 2 (CS2) by residues R31 and K32 located at the HTH-1 domain. f, Interactions between T92 and K95 (HTH-2 domain) and the CS1 of donor DNA.

Supplementary Fig. 6 | Effect of IstA mutants on the integration reaction.
Representative native agarose gel of the effect of the catalytic and DNA-binding mutants used in this study. This experiment was repeated four times (except for E233A that was repeated three times). Source data are provided as a Source Data file. a, Alignment of the four repeats (L1, L2, R1 and R2) reveals the presence of two conserved sequence motifs (CS1 and CS2). These regions are contacted by the HTH-1 and HTH-2 domains. b, The HTH domains of the catalytic and non-catalytic IstA monomers interact with the multiple terminal repeats (R1 and R2, respectively) using similar protein-DNA contacts. The left transposon end was not used in the highresolution structure but, due to the elevated sequence identity that exists between the left and right transposon ends, the interaction with the L1 and L2 repeats (marked with asterisk) is likely established using similar contacts.  ]) are separated by 44 Å and also produce staggered cuts that generate 5 bp repeats. d, Ideal linear target DNA modelled in the active site shows how the position of the IstA catalytic domains would generate a pair of cuts staggered ~2 bp apart (indicated with black segment) and, therefore, are offset from the positions needed to produce the characteristic 5 bp direct duplications of this element (for comparison 5 bases are colored in yellow). e, TnsB target DNA modelled on IstA shows that the configuration of the DDE motifs would generate shorter staggered cuts and, consequently, direct repeats (compare black segment with that shown in b). f, MuA target DNA modelled on IstA shows that the configuration of the DDE motifs would also generate shorter staggered cuts and direct repeats (compare black segment with that shown in c).

Supplementary Tables
Supplementary Table 1. Oligonucleotides used for the functional, biochemical and structural assays. Terminal repeats are underlined and catalytic adenosine colored red the first they appear in the table and for the cryo-EM substrates. Name Sequence ( Fig. 1g. Substrates were obtained as follow:
Left and right end pre-cleaved duplexes with similar overhang configurations were mixed in an equimolar ratio (1 µM total). Supplementary  Fig. 2b.
L-TIR-rev was annealed with L-TIR-Over5bp-fwd to generate a pre-cleaved substrate with a 5-base long 5'overhang.
R-TIR-fwd was annealed with R-TIR-Over5bp-rev to generate a pre-cleaved substrate with with a 5-base long 5'overhang.
Left and right end pre-cleaved duplexes were mixed in an equimolar ratio (1 µM total). Pre-cleaved right TIR duplex for structural studies was generated annealing R-TIR-fwd with R-TIR-Over5bp-rev.